Adeno-associated virus (AAV) is a dependent parvovirus that integrates into a specific locus (the preintegration site) of the human genome. This unique feature of AAV makes it particularly attractive as a gene therapy vector. Targeted integration of wild type and recombinant (r) AAV requires the virally encoded Rep proteins. While these proteins are essential for targeted integration, Rep has also been shown to be required for AAV viral replication, to auto-regulated its own promoter, and to transactivate and repress AAV and other heterologous gene cassettes. However, constitutive or low level expression of Rep can also have cytotoxic effects on cells. To address this conundrum, the role of Rep in target integration must be better understood. This will require studies to dissect Rep-mediated integration from its other activities. The primary objective of this proposal is to determine the mechanism of Rep-mediated targeted integration by using temperature sensitive and specific Rep mutant proteins. The specific goals of this project are divided into two aims: 1) to develop and characterize temperature sensitive Rep proteins (tsRep), and other specific Rep mutants, and determine their ability to facilitate targeted integration; and 2) to characterize the reaction parameters required for targeted integration mediated by the tsRep proteins. To accomplish these aims, targeted integration will be analyzed at permissive and non-permissive temperatures by co-transfection experiments using modified AAV plasmids and a human cell line carrying the pre-integration site on a episome vector. These experiments will establish the parameters of Rep-mediated targeted integration and determine the feasibility of using tsRep mutants to generate targeting vectors. The long term objective is to optimize targeted integration of rAAV vectors for gene therapy.